1. Field of the Invention
The present invention relates generally to the fields of the molecular biology of T cell signaling and fatty acid biochemistry and pharmacology. More specifically, the present invention relates to novel uses of 2-bromopalmitate.
2. Description of the Related Art
Many viral and cellular proteins are modified by fatty acid acylation with myristate or palmitate (1,2). For example, all members of the Src family of tyrosine protein kinases are covalently modified by the 14 carbon fatty acid myristate. Myristate is co-translationally attached to a glycine at position 2 of the protein through an amide linkage, in a process catalyzed by N-myristoyl transferase (NMT) (35). Myristoylation has been shown to be necessary (6,7) but not sufficient (8) for membrane binding. In addition, all Src proteins use a second membrane targeting signal. For seven out of the nine Src family members, this second signal involves modification with the 16 carbon fatty acid palmitate. Palmitate is post-translationally attached to a cysteine residue within an N-terminal myr-gly-cys consensus motif (9).
Attachment of myristate and palmitate to Src family kinases enhances the localization of these proteins to the plasma membrane, where they must be present in order to function properly. In addition, protein palmitoylation has been shown to be critical for localization of proteins to specialized subdomains of the plasma membrane that are resistant to detergent extraction (10-15). These detergent resistant microdomains (detergent resistant microdomains), also known as rafts, are enriched in cholesterol, glycosphingolipids, and GPI-anchored proteins (16-18). Localization to detergent resistant microdomains influences the ability of key signaling molecules to interact with each other and to participate in signaling from the cell surface to the interior of the cell (11,19-21).
The importance of protein fatty acylation is best illustrated 0.5 in T cell receptor (TCR) mediated signal transduction. The Src related kinases Fyn and Lck are highly expressed in cells of hematopoietic origin, particularly lymphocytes (22), and are required for signaling through the T cell receptor. Protein tyrosine phosphorylation is one of the first events that occurs after binding of antigens to surface receptors in T lymphocytes. Upon receptor engagement, Fyn and Lck phosphorylate tyrosine residues found within multiple immunoreceptor tyrosine-based activation motifs (ITAMS) located on the cytosolic portions of the TCRζ and CD3 chains. Immunoreceptor tyrosine-based activation motifs phosphorylation recruits key molecules that mediate downstream signaling, including the tyrosine kinase ZAP-70 (19). One of the targets for activated ZAP-70 is LAT, a palmitoylated transmembrane protein (10). Several recent studies have established that the ability of Lck, Fyn and LAT to function in T cell receptor-mediated signaling depends on their fatty acylation and localization to detergent resistant microdomains. Palmitoylation of Lck was shown to be essential for its signaling function in T lymphocytes (11). Fyn must be palmitoylated and localized to detergent resistant microdomains in order to interact with the ζ chain of the T cell receptor (23). Moreover, LAT must be palmitoylated and in detergent resistant microdomains in order to become tyrosine phosphorylated and participate in downstream signaling (20).
To date, studies of the role of protein palmitoylation in various cellular pathways have suffered from two major drawbacks. First, in contrast to N-myristoylation, very little is known about the enzymology and biochemistry of protein palmitoylation. Two thioesterases, PPT1 and AP1, have been identified that deacylate palmitoylated Ras and Gα proteins in vitro (24,25). However, the enzyme(s) that catalyze(s) attachment of palmitate to proteins have not been definitively identified. Several recent studies have described purification of palmitoyl acyl transferase (PAT) activities (26-28), while other reports have documented that non-enzymatic palmitoylation can occur under certain conditions in vitro (29,30). Second, nearly all studies reported to date on the role of palmitoylation in cellular functions have been limited to expressing non-acylated mutant forms of proteins in various systems (11,20). While this approach does provide useful information, it is limited by the need to overexpress the mutant proteins. Furthermore, the loss of a cysteine residue, and not the loss of palmitate per se, may impair the ability of the protein to function properly. For example, Hepler et al showed that cysteine residues at the amino terminus of the Gq alpha subunit is important for its interaction with effector and receptor molecules, regardless of their state of palmitoylation (31).
Polyunsaturated fatty acids (PUFAs), particularly the n-3 series, are used clinically as immunosuppressive agents (32) and in the treatment of various inflammatory diseases (33-36). Recently, it was reported that polyunsaturated fatty acids inhibit T cell signal transduction by displacing Fyn and Lck from the detergent resistant microdomains (37). The inhibitory effects of polyunsaturated fatty acids were hypothesized to be mediated by modification of DRM structure and composition.
The prior art is deficient in the lack of specific inhibitors of Fyn and Lck fatty acylation and protein palmitoylation. The present invention fulfills this longstanding need and desire in the art.